The integrin α2β1 (Very late antigen 2; VLA-2) is expressed on a variety of cell types including platelets, vascular endothelial cells, epithelial cells, activated monocytes/macrophages, fibroblasts, leukocytes, lymphocytes, activated neutrophils and mast cells. (Hemler, Annu Rev Immunol 8:365:365-400 (1999); Wu and Santoro, Dev. Dyn. 206:169-171 (1994); Edelson et. al., Blood. 103(6):2214-20 (2004); Dickeson et al, Cell Adhesion and Communication. 5: 273-281 (1998)). The most typical ligands for α2β1 include collagen and laminin, both of which are found in extracellular matrix. Typically the I-domain of the α2 integrin binds to collagen in a divalent-cation dependent manner whereas the same domain binds to laminin through both divalent-cation dependent and independent mechanisms. (Dickeson et al, Cell Adhesion and Communication. 5: 273-281 (1998)) The specificity of the α2β1 integrin varies with cell type and serves as a collagen and/or laminin receptor for particular cell types, for example α2β1 integrin is known as a collagen receptor for platelets and a laminin receptor for endothelial cells. (Dickeson et al, J. Biol. Chem. 272: 7661-7668 (1997)) Echovirus-1, decorin, E-cadherin, matrix metalloproteinase I (MMP-I), endorepellin and multiple collectins and the C1q complement protein are also ligands for α2β1 integrin. (Edelson et al., Blood 107(1): 143-50 (2006)) The α2β1 integrin has been implicated in several biological and pathological processes including collagen-induced platelet aggregation, cell migration on collagen, cell-dependent reorganization of collagen fibers as well as collagen-dependent cellular responses that result in increases in cytokine expression and proliferation, (Gendron, J. Biol. Chem. 278:48633-48643 (2003); Andreasen et al., J. Immunol. 171:2804-2811 (2003); Rao et al., J. Immunol. 165(9):4935-40 (2000)), aspects of T-cell, mast cell, and neutrophil function (Chan et. al., J. Immunol. 147:398-404 (1991); Dustin and de Fougerolles, Curr Opin Immunol 13:286-290 (2001), Edelson et. al., Blood. 103(6):2214-20 (2004), Werr et al., Blood 95:1804-1809 (2000), aspects of delayed type hypersensitivity contact hypersensitivity and collagen-induced arthritis (de Fougerolles et. al., J. Clin. Invest. 105:721-720 (2000); Kriegelstein et al., J. Clin. Invest. 110(12):1773-82 (2002)), mammary gland ductal morphogenesis (Keely et. al., J. Cell Sci. 108:595-607 (1995); Zutter et al., Am. J. Pathol. 155(3):927-940 (1995)), epidermal wound healing (Pilcher et. al., J. Biol. Chem. 272:181457-54 (1997)), and processes associated with VEGF-induced angiogenesis (Senger et al., Am. J. Pathol. 160(1):195-204 (2002)).
Integrin/ligand interactions can facilitate leukocyte extravasation into inflamed tissues (Jackson et al., J. Med. Chem. 40:3359-3368 (1997); Gadek et al., Science 295(5557):1086-9 (2002), Sircar at al., Bioorg. Med. Chem. 10:2051-2066 (2002)), and play a role in downstream events following the initial extravasation of leukocytes from the circulation into tissues in response to inflammatory stimuli, including migration, recruitment and activation of pro-inflammatory cells at the site of inflammation (Eble J. A., Curr. Phar. Des. 11(7):867-880 (2005)). Some antibodies that block α2β1 integrin were reported to show impact on delayed hypersensitivity responses and efficacy in a murine model of rheumatoid arthritis and a model of inflammatory bowel disease (Kriegelstein et al., J. Clin. Invest. 110(12):1773-82 (2002); de Fougerolles et. al., J. Clin. Invest. 105:721-720 (2000) and were reported to attenuate endothelial cell proliferation and migration in vitro (Senger et al., Am. J. Pathol. 160(1):195-204 (2002), suggesting that the blocking of α2β1 integrin might prevent/inhibit abnormal or higher than normal angiogenesis, as observed in various cancers.
α2β1 integrin is the only collagen-binding integrin expressed on platelets and has been implicated to play some role in platelet adhesion to collagen and hemostasis (Gruner et al., Blood 102:4021-4027 (2003); Nieswandt and Watson, Blood 102(2):449-461 (2003); Santoro et al., Thromb. Haemost. 74:813-821 (1995); Siljander et al., Blood 15:1333-1341 (2004); Vanhoorelbeke at al., Curr. Drug Targets Cardiovasc. Haematol. Disord. 3(2):125-40 (2003)). In addition, platelet α2β1 may play a role in the regulation of the size of the platelet aggregate (Siljander et al., Blood 103(4):1333-1341 (2004)).
α2β1 integrin has also been shown as a laminin-binding integrin expressed on endothelial cells (Languino et al., J Cell Bio. 109:2455-2462 (1989)). Endothelial cells are thought to attach to laminin through an integrin-mediated mechanism, however it has been suggested that the α2 I domain may function as a ligand-specific sequence involved in mediating endothelial cell interactions (Bahou et al., Blood. 84(11):3734-3741 (1994)).
It is anticipated that a therapeutic antibody that binds α2β1 integrin, including the α2β1 integrin on platelets, could result in bleeding complications. For example, antibodies targeting other platelet receptors such as GPIb (Vanhoorelbeke et al., Curr. Drug Targets Cardiovasc. Haematol. Disord. 3(2):125-40 (2003) or GP IIb/IIIa (Schell at al., Ann. Hematol. 81:76-79 (2002), Nieswandt and Watson, Blood 102(2):449-461 (2003), Merlini et al., Circulation 109:2203-2206 (2004)) have been associated with thrombocytopenia, although the mechanisms behind this are not well understood. It has been hypothesized that binding of an antibody to a platelet receptor can alter its three dimensional structure, and expose normally unexposed epitopes which then leads to platelet elimination (Merlini et al., Circulation 109:2203-2206 (2004). Indeed, the bleeding complications associated with oral doses of GP IIa/IIIb antagonists have been described as the “dark side” of this class of compounds (Bhatt and Topol, Nat. Rev. Drug Discov. 2(1):15-28 (2003)).
The anti-human α2β1 integrin blocking antibody BHA2.1 was first described by Hangan et al., (Cancer Res. 56:3142-3149 (1996)). Other anti-α2β1 integrin antibodies are known and have been used in vitro, such as the commercially available antibodies AK7 (Mazurov et al., Thromb. Haemost. 66(4):494-9 (1991), P1E6 (Wayner et al., J. Cell Biol. 107(5):1881-91 (1988)), 10G11 (Giltay et al., Blood 73(5):1235-41 (1989) and A2-11E10 (Bergelson et al., Cell Adhes. Commun. 2(5):455-64 (1994). Hangan et al., (Cancer Res. 56:3142-3149 (1996)) used the BHA2.1 antibody in vivo to study the effects of blocking α2β1 integrin function on the extravasation of human tumor cells in the liver, and the ability of these tumor cells to develop metastatic foci under antibody treatment. The Ha1/29 antibody (Mendrick and Kelly, Lab Invest. 69(6):690-702 (1993)), specific for rat and murine α2β1 integrin, has been used in vivo to study the upregulation of α2β1 integrin on T cells following LCMV viral activation (Andreasen et al., J. Immunol. 171:2804-2811 (2003)), to study SRBC-induced delayed type hypersensitivity and FITC-induced contact type-hypersensitivity responses and collagen-induced arthritis (de Fougerolles et. al., J. Clin. Invest. 105:721-720 (2000)), to study the role of α2β1 integrin in VEGF regulated angiogenesis (Senger et al., Am. J. Pathol. 160(1):195-204 (2002); Senger et al., PNAS 94(25): 13612-7 (1997)), and to study the role of α2β1 integrin in PMN locomotion in response to platelet activating factor (PAF) (Werr et al., Blood 95:1804-1809 (2000)).
The use of murine monoclonal antibodies, such as those described above, as human therapeutic agents in non-immunocompromized patients has been limited by the robust immune responses directed against administered murine antibodies, particularly in repeated administration. This response cannot only curtail the effective half-life of the murine antibody in circulation but also can lead to profound injection site and/or anaphylactic responses (Shawler et al., J. Immunol. 135(2):1530 (1985)). In addition, the rodent effector functions associated with the constant regions (Fc) are much less effective than their human counterparts when administered to humans, resulting in a loss of potentially desirable complement activation and antibody-dependent, cell-mediated cytotoxicity (ADCC) activity.